title: Ryanodine receptor phosphorylation by oxidized CaMKII contributes to the cardiotoxic effects of cardiac glycosides.
abstract: OBJECTIVE
  Recent studies suggest that proarrhythmic effects of cardiac glycosides (CGs) on cardiomyocyte Ca(2+) handling involve generation of reactive oxygen species (ROS). However, the specific pathway(s) of ROS production and the subsequent downstream molecular events that mediate CG-dependent arrhythmogenesis remain to be defined.
  
  
  RESULTS
  We examined the effects of digitoxin (DGT) on Ca(2+) handling and ROS production in cardiomyocytes using a combination of pharmacological approaches and genetic mouse models. Myocytes isolated from mice deficient in NADPH oxidase type 2 (NOX2KO) and mice transgenically overexpressing mitochondrial superoxide dismutase displayed markedly increased tolerance to the proarrhythmic action of DGT as manifested by the inhibition of DGT-dependent ROS and spontaneous Ca(2+) waves (SCW). Additionally, DGT-induced mitochondrial membrane potential depolarization was abolished in NOX2KO cells. DGT-dependent ROS was suppressed by the inhibition of PI3K, PKC, and the mitochondrial KATP channel, suggesting roles for these proteins, respectively, in activation of NOX2 and in mitochondrial ROS generation. Western blot analysis revealed increased levels of oxidized CaMKII in WT but not in NOX2KO hearts treated with DGT. The DGT-induced increase in SCW frequency was abolished in myocytes isolated from mice in which the Ser 2814 CaMKII phosphorylation site on RyR2 is constitutively inactivated.
  
  
  CONCLUSIONS
  These results suggest that the arrhythmogenic adverse effects of CGs on Ca(2+) handling involve PI3K- and PKC-mediated stimulation of NOX2 and subsequent NOX2-dependent ROS release from the mitochondria; mitochondria-derived ROS then activate CaMKII with consequent phosphorylation of RyR2 at Ser 2814.
authors.affiliation: Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH, USA.
authors.forename: Hsiang-Ting
authors.initials: HT
authors.lastname: Ho
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
authors.forename: Jedidiah S
authors.initials: JS
authors.lastname: Snyder
authors.forename: Qing
authors.initials: Q
authors.lastname: Lou
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Brundage
authors.forename: Florencia
authors.initials: F
authors.lastname: Velez-Cortes
authors.forename: Honglan
authors.initials: H
authors.lastname: Wang
authors.forename: Mark T
authors.initials: MT
authors.lastname: Ziolo
authors.forename: Mark E
authors.initials: ME
authors.lastname: Anderson
authors.forename: Chandan K
authors.initials: CK
authors.lastname: Sen
authors.forename: Xander H T
authors.initials: XH
authors.lastname: Wehrens
authors.forename: Vadim V
authors.initials: VV
authors.lastname: Fedorov
authors.forename: Brandon J
authors.initials: BJ
authors.lastname: Biesiadecki
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Hund
authors.forename: Sándor
authors.initials: S
authors.lastname: Györke
mesh_terms: D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000633:toxicity*; D001145:Arrhythmias, Cardiac / Q000139:chemically induced; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D004074:Digitoxin / Q000633:toxicity*; D008297:Male; D053078:Membrane Potential, Mitochondrial; D051379:Mice; D018345:Mice, Knockout; D032383:Myocytes, Cardiac / Q000187:drug effects*; D019255:NADPH Oxidases / Q000378:metabolism; D010766:Phosphorylation; D017382:Reactive Oxygen Species / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism
chemical_list: D000889:Anti-Arrhythmia Agents; D017382:Reactive Oxygen Species; D019837:Ryanodine Receptor Calcium Release Channel; D004074:Digitoxin; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D002118:Calcium
keywords: CaMKII; Calcium; Mitochondria; NADPH oxidase; Reactive oxygen species